To culture cells in vitro, most of nutrients and growth factors that are necessary for culture should be artificially put into a culture medium solution. By taking in the nutrients and growth factors that are dissolved in the culture medium solution, cultured cells keep themselves alive, proliferate, and produce useful substances. During that time of period, the cells consume e.g., oxygen, nutrients, and growth factors, and excrete, e.g., carbon dioxide and waste matters including metabolites. In the case where cells are cultured for a long time of period, the amount of nutrients in a culture medium solution or oxygen in surroundings may become to be insufficient and thus the cells may become not to proliferate or exist.
By the way, when an influence of a medicine, a growth factor, an inhibitor, a regulator, a toxin, a unknown factor, a nutrient, a chemical, or the like on cells is studied, a relationship between, not the absolute amount of the medicine but the concentration of the medicine in a culture medium solution, and the influence of its presence on the cells, is studied. In this case there is a problem that, as a culturing time elapses, the concentration of the medicine in the culture medium solution lowers because the medicine is consumed, namely, the concentration of the medicine cannot be constantly maintained.
Conventionally, in the case where cells or a piece of a tissue are cultured for a long time of period and influences of a medicine on them are studied or observed by using, as a container for culture, a petri dish, a flask, a multi-well plate, or the like, in the beginning of the culture a small number of the cells or a small amount of a tissue is used and a large amount of a culture medium solution is put into the container for culture, or the culture medium solution is exchanged during the culture. Now, we explain more specifically about the latter case. Cells or a piece of a tissue should be studied or observed in a certain cell density or a certain amount of the tissue that is suitable for culture. Also, according to the cell density or the amount of the tissue, a suitable amount of the culture medium solution may be specified. Therefore, the culture experiment may be started by using a suitable amount of a culture medium solution containing a medicine to be examined in a predetermined concentration, and if the period of studying or observing the cells or the piece of the tissue is prolonged, it is necessary that all or part of the culture medium solution is exchanged with a fresh and same solution containing the medicine to be examined in the same, i.e., initial concentration.
Conventional methods, by which cells or a piece of a tissue (hereafter, “cells or the like”) are cultured by using, as a container, a petri dish, a flask, a multi-well plate, or the like and by which the cells or the like are studied or observed for a long time of period, have the following defects:
(1) In some cases the culture should be started under such a condition that the number of cells or the amount of a tissue is small in a comparison with the amount of the culture medium solution, that is, the density of the cells or the like is low. That the cell density or the like is low usually means that the surroundings or environment for proliferation are poor. Therefore, the start of the logarithmic growth phase lags, in other words, the preparatory period for proliferation becomes long.
(2) If a large amount of a culture medium solution is used in a container (this is useful to minimize a fluctuation of a concentration of a medicine to be examined), the level of the culture medium solution becomes high. In this case an efficiency of exchange of gases such as oxygen and carbon dioxide in the bottom (where the cells adhere) of the container for culture is low, namely, culturing conditions are poor.
(3) During an exchange of a culture medium solution, it is necessary to conduct an additional axenic manipulation, a risk of contamination is increased, it is difficult to hold the amount of the culture medium solution to be constant after its exchange, and the cells and the like tend to move from their original positions to other positions after the exchange of the culture medium solution, except that cells have a property that they adhere or stick tight to a container. Therefore, exchange of the culture medium solution during culture is disadvantageous to study or observe the same cells continuously.
Now, we more specifically explain the above defect 1. In a logarithmic growth phase, the time that the number of cells becomes twice the original number of them by their fissions is constant. Therefore, until, e.g., contact inhibition arises, the number of cells becomes 2n after the above-mentioned constant time runs n-times. If the contact inhibition and the like does not arise, the cells continuously proliferate while, in some cases, layering to each other as long as good conditions are maintained about nutrients, oxygen, waste matters, and the like. If an influence of a medicine or the like on cells is studied during the logarithmic growth phase, the influence can be readily recognized. However, if the cell density is low in a culture medium solution or if culturing conditions such as medium components differ from those in preliminary culture, usually cells do not come to a logarithmic growth phase right after culture is started, although it depends on the kind and character of cells. Further, if the cell density is low, it takes a longer time of period to change culture surroundings to be convenient for proliferation of cells by producing growth factors and the like by which the cells are up-regulated. For conditioning the surroundings, it is advantageous that the same kind of cells are nearby gathered, but there are disadvantageous that the number of cells is too small and that the cells are sparse. Because of the reasons stated above, the leading or warm-up time, i.e., a period until a study of an influence of a medicine can be started, becomes longer.
Therefore, for example, when myeloma cells that proliferate rapidly are cultured for a relatively long period of time, i.e., for five days or longer, and with the lapse of time an influence of a medicine on the myeloma cells is studied, a method wherein a large amount of a culture medium solution is used, a method wherein a small number of cells is innoculated, or both are usually conducted. However, when these methods are conducted, the cell density becomes low. If one tries to study or observe states of cells under a condition that the cell density is low, only a few number of cells can be observed in a view field of a microscope under a magnification that the states of cells can be checked. Thus, one can not efficiently observe the cells. If the cell density is adequate for observation at the beginning of culture, in other words, if culture is started under a condition that the cell density is high, it is difficult to study or observe with the lapse of time the influences of a medicine to be examined, a nutrient, or the like on the cells for a long period of time without exchange of the culture medium solution. This is because the medicine to be examined, nutrient, or the like in the culture medium solution is rapidly consumed.
There are some apparatuses by which cell density can be locally increased to some extent. For example, in round (U-shape) bottomed or V-shape bottomed micro-titer plates, cells can be concentrated near the center bottom of wells. This is because the cells are settled down by their gravity. Thus, one can start to culture the cells at a high cell density locally with relatively a larger amount of a culture medium solution. However, the use of the round (U-shape) bottomed or V-shape bottomed micro-titer plates have following defects:
(1) The cells may excessively contact to each other. Thus, it is difficult to use the plates to culture cells that suffer contact inhibition.
(2) The cells may make laminated layers. For observing the cells by using a microscope, it is inconvenient if they exist in different vertical positions. For observing the cells during their culture, they are better that the bottom of a well or a container is substantially flat in horizontal direction (e.g., flat bottom or C-shape bottom) and that the cells exist as a monolayer.
(3) When cells become a multiple layer, oxygen, nutrients, etc., may come short in the lower layer.
(4) If a micro-titer plate is used, the amount of a culture medium solution in a well may not be increased. Even if the well is filled with the culture medium solution, its amount is, e.g., 0.3 milliliter, and would still be small to culture cells for a long time of period. Thus, in the course of culture, the solution should be exchanged. However, when the solution is exchanged, the cells may disperse. To avoid the exchange of the culture medium solution, a micro-titer plate having deep wells may be used. However, if the deep well is filled with the culture medium solution, the depth of it is increased. In this case, the culturing conditions would become poor. For example, oxygen will become short at the bottom where cells exist. This is because oxygen derived from air phase difficultly reaches to the bottom.
(5) To condition the environment for proliferation, cells would produce up-regulating factors and the like. If the volume of the culture medium solution is simply increased, the factors would quickly diffuse or spread in the culture medium solution. Namely, the factors would be diluted. This is not preferable. It is better that the cell-produced up-regulating factors accumulate near the cells.
Followings are other known methods for culturing cells and the like in vitro. Namely, Japanese Patent Publication No. Hei. 06-44860 B discloses a method wherein a spinner bottle and an insert are used, at least of which insert is constituted by a cell-impermeable and liquid medium-permeable net or membrane, and culture is conducted while continuously exchanging a culture medium solution inside the insert for a culture medium solution outside the insert through the net or membrane. Japanese Patent Publication No. Hei. 07-97982 B discloses a method for obtaining a cell product by culturing cells for a long time of period outside hollow fibers while supplying nutrients and oxygen to the cells by circulating a culture medium solution in the hollow fibers. Japanese Patent No. 2619885 discloses a method wherein cells are trapped inside hollow fibers, substances that are essential to culture the cells are supplied by circulating a liquid such as a culture medium solution outside the hollow fibers, and metabolites that the cells secrete and that are dissolved in the liquid are isolated from the liquid. However, these methods were developed for the purpose of culturing a larger number of cells or simplifying the isolation of cells' metabolites or products. Thus, these methods are not suitable to study or observe states of cells with the lapse of time.
Apparatuses have also been proposed that are suitable to culture cells and to study states of the cells with the lapse of time. For example, an apparatus that is used by combining a tissue culture insert (Nalge Nunc International) with a multi dish (Nalge Nunc International) and other apparatus that is used by combining a cell culture insert (Becton Dickinson and Company) with a companion plate (Becton Dickinson and Company) are known. To these apparatuses the principle of the invention disclosed in U.S. Pat. No. 4,308,351, namely, a method wherein an apparatus comprising a well and an insert is used which insert comprises a permeable membrane in its upper part, and a tissue is cultured in the insert (i.e., under a permeable membrane), is applied.
Further, European Patent Publication Nos. 0638640 A2 and 0590513 A2 disclose apparatuses that are useful when an interaction between two kinds of cells is studied without physically contacting two groups of the cells to each other by separating the groups by a membrane. They were invented by developing the invention disclosed in U.S. Pat. No. 4,308,351. In the apparatuses disclosed in European Patent Publication Nos. 0638640 A2 and 0590513 A2, an insert is put into a culture vessel wherein the bottom of the insert is made of a microporous membrane through which cells can not pass. The cells are cultured on and below the membrane, namely, in two places which are within the culture vessel and within the insert. In these apparatuses, a culture medium solution is supplied to the inside of the insert through the membrane. Also, the culture medium solution can be exchanged after the insert is taken out from the vessel. Further, in the apparatus disclosed in European Patent Publication No. 0590513 A2, a culture medium solution can be exchanged through a pipette that has been inserted into the culture medium solution between an inner wall of the vessel and an outer wall of the insert. Therefore, when these apparatuses are used, it is not necessary to heighten the level of the culture medium solution so much. Further, these apparatuses have a structure that there is a space on and above the surface of the culture medium solution and that therefore an enough amount of gases can be supplied to the cells. By using these apparatuses, the states of the cells can also be studied by taking out the insert from the culture vessel.
Japanese Patent Publication No. Hei. 07-46988 B discloses a technique by which cells are cultured using a porous material that is holding a culture medium solution. Specifically, it discloses that a culture medium solution is held by a porous polyurethane foam (PUF) in which polyurethane molecules a matrix of a peptide such as collagen is made and that adhesive animal cells are adhered to the foam and are cultured. In this invention, the PUF has a form of a chip having a size of preferably 1 to 3 mm and plays as a carrier to which the cells are adhered. Nutrients and gases that are necessary to culture the cells are supplied from a culture medium solution inside a culture vessel, into which the PUF chips are put.
One example of materials that can be gelatinized is agar. As a method for culturing cells by using agar, a soft agar method has been known, which is used for cloning and was published about 30 years ago (please see R. G. H. Cotton, et. al., Eur. J. Immunol., 3, p.p. 135–140 (1973) and John Paul, Cell and Tissue Culture, 4th Ed., Churchill Livingstone Edinburgh and London, p.p. 234–239 (1973)). This method comprises seeding cells in an extremely low density in a culture medium solution containing agar in a low concentration and culturing in gelatinous soft agar cell colonies, each of which has proliferated from one cell. Also, in assays for blood stem cells or myeloid stem cells, colonies are formed in soft agar or methylcellulose or its derivative and are studied or observed.
Further, in experiments using cultured cells, electric potentials of the cells are often measured. Examples thereof include methods wherein electric potentials of cells are measured by contacting the cells with electrodes that are placed on a bottom of a container (please see Japanese Patent Early-publication Nos. Hei. 06-296595 and Hei. 08-62209). However, if the soft agar method is used in these methods, it becomes difficult to contact the cells with the electrodes. Thus, the soft agar method is disadvantageous for measuring electric potentials of cultured cells.
A method has been known wherein an electrical resistance that arises in epithelial cells when an electric current passes through mono-layered those cells is measured while culturing those cells without using the soft agar method. In that method, using an apparatus disclosed in U.S. Pat. No. 4,686,190, an electric current is passed through a layer that has been formed by fusing a support with cells that has been proliferated on the support.